Antiviral combinations

ABSTRACT

Antiviral combinations containing a 2-ester-substituted-3,4-dihydro-3-oxoquinoxaline and a hindered amine.

This is a division, of application Ser. No. 95,804 filed Nov. 19, 1979,which was a division of my copending application Ser. No. 49,671, filedJune 18, 1979, now U.S. Pat. No. 4,210,647, issued July 1, 1980.

BACKGROUND OF THE INVENTION

2-Ester-substituted-3,4-dihydro-3-oxo-quinoxalines are described asantiviral agents in copending application Ser. No. 953,157, filed Oct.20, 1978, now abandoned, in Ser. No. 60,445 filed July 25, 1979, nowabandoned, and in Ser. No. 88,274, filed Oct. 25, 1979. The compoundsare particularly useful in that they show antiviral activity againstboth A and B strains of influenza virus. The antiviral activity of1-aninoadamantane (amantidine, adamantylamine) was first disclosed byDavies et al. Science, 144, 862 (1964). 1-aminoadamantane is said to beactive against influenza virus A² strain prophylactically. The compoundalso may have some use in the prophylaxis of Asian flu strainsantigenically related to strain A². 3-Methyl-1-aminoadamantane,dl-cyclooctylamine, 2-norbornylamine and 1-adamantyl-1-aminoethane(rimantadine) have all been shown to be antiviral agents, both by myselfand those working with me and by those working in other laboratories.

SUMMARY OF THE INVENTION

This invention provides a combination of a2-ester-substituted-3,4-dihydro-3-oxoquinoxaline of the formula ##STR1##wherein each X is separately Cl or Br and R is methyl or ethyl plus ahindered amine selected from the group consisting of 1-aminoadamantane,(formula II below when R¹ is NH₂ and R₂ is H) 3-methyl-1-aminoadamantane(formula II below wherein R¹ is NH₂ and R² is methyl), 1adamantyl-1-aminoethane (formula II below wherein R¹ is and R² is CH₃ orH), dl-cyclooctylamine and norbornanamine also referred to as1-aminonorbornane and 2-aminobicyclo[2.2.1]-heptane (formula III below).##STR2##

The above combination of antiviral drugs can be administered bothprophylactically and therapeutically; i.e., both pre and post-infection.The combination can thus be given to mammals exposed to influenza virusto abort the viral infection as well as to mammals who have contractedan influenzal infection and are in need of a viral growth suppressingagent. Whether given before or after viral infection is present, theabove combination will suppress the growth of both A and B strains ofinfluenza virus. It is a particular advantage of my novel antiviral drugcombination that a determination of the strain of influenza viruscausing a particular infection need not be made since the combination iseffective against both A and B strains.

The customary dose levels used in my novel antiviral combination varywidely depending on weight, size, body surface etc. of the particularmammalian species involved. With mice, for example, a single dose willcontain from 20 to 160 mg./kg. of a quinoxaline according to formula Iabove plus from 10-80 mg./kg. of 1-aminoadamantane or from 5-50 mg./kg.of 3-methyl-1-aminoadamantane or 10-80 mg./kg. each ofdl-cyclooctylamine, 2-aminonorbornane, or 1-(1-adamantyl)-1-aminoethane.For humans, the dose level is from 1/10 to 1/50 that of the dosage formice, i.e., from 0.5-5 mg./kg. of a quinoxaline according to formula Iplus 0.3-3 mg./kg. of 1-aminoadamantane or about 0.2-2 mg./kg. of3-methylaminoadamantane or 0.3-3 mg./kg. of dicyclooctylamine,2-aminonorbornane or 1-(1-adamantyl)-1-aminoethane. An average dailydosage schedule for humans would be from 30-300 mg. of a quinoxalineplus 15-200 mg. of 1-aminoadamantane twice a day. Dosage schedules forother combinations can be derived from the above data, coupled with aknowledge of dosage variation according to mammalian species with otherdrugs. The dosage form containing a quinoxaline of formula I plus ahindered amine containing drug-weights coming within the above ranges,is usually administered for four days consecutively although dosages of1, 2 or 3 day duration will also provide good results as regards theviral infection.

Quinoxalines according to formula I above are prepared by reacting a4,5-dihalophenylenediamine with a dialkyl mesoxalate according to thefollowing reaction scheme ##STR3## wherein R and X have the previouslyassigned significance. The following examples illustrate the procedureof Reaction Scheme I for preparing compound according to formula Iabove.

EXAMPLE 1 Preparation of Ethyl6,7-Dichloro-3,4-dihydro-3-oxo-2-quinoxaline Carboxylate.

A solution of 17.7 g. of 4,5-dichloro-o-phenylenediamine in 200 ml. ofanhydrous ethanol was prepared. A 17.4 g. batch of diethyl2-ketomalonate were added to this solution and the mixture was heated torefluxing temperature for about 17 hours. The volatile constituents wereremoved by evaporation in vacuo. Recrystallization of the residue fromethanol yielded 20 g. of ethyl6,7-dichloro-3,4-dihydro-3-oxo-2-quinoxaline carboxylate formed in theabove reaction, melting in the range 226°-227° C.

EXAMPLE 2 Preparation of Ethyl 6,7-dibromo-2,3-dihydro-3-oxoquinoxalineCarboxylate.

Ten g. of 3,4-dibromoaniline were mixed with 40 ml. of acetic anhydride.The resulting reaction mixture was heated to a temperature in the range100°-105° C. for one hour after which time it was poured over a mixtureof ice and water. After stirring overnight, the aqueous mixture yieldedan off-white precipitate weighing 11.5 g. and melting at 90°-95° C.comprising 3,4-dibromoacetanilide.

2.5 g. of 3,4-dibromoacetanilide were mixed with 8 ml. of 18 N aqueoussulfuric acid at 0° C. 1.5 g. of propyl nitrate were added whilemaintaining the reaction temperature in the range of 0°-2° C. Thechilled reaction mixture was stirred for one hour at the sametemperature and then poured over an ice-water mixture. A yellow solidcomprising 3,4-dibromo-6-nitroacetanilide formed in the above reactionprecipitated and was collected by filtration. Recrystallization fromethanol yielded 1.2 g. of 3,4-dibromo-6-nitroacetanilide melting at140°-141° C. after recrystallization from ethanol.

One gram of 3,4-dibromo-6-nitroacetanilide was heated to refluxingtemperature for 30 minutes with 30 ml. of 6 N aqueous hydrochloric acid.The reaction mixture was then poured over an ice-water mixture withstirring. The pH of the solution was adjusted to 12 with alkali. Theresulting bright yellow precipitate was separated by filtration, washedand dried; yield=0.85 g of 3,4-dibromo-6-nitroaniline melting at204°-205° C.

Five grams of 3,4-dibromo-6-nitroaniline were suspended in 200 ml. ofanhydrous ethanol to which was added about 10 g. of Raney nickel. Thehydrogenation mixture was placed in a low pressure hydrogenationapparatus at a hydrogen pressure of 55 psi. A rapid uptake of hydrogenoccurred which ceased after about 25 minutes, at which time the deepyellow color originally present had been discharged indicating completereduction of the nitro group to an amino group. The hydrogenation wascontinued for another half hour and the hydrogenation mixture was thenworked up by filtering off the catalyst, washing the filtered catalyst,and stripping the volatile constituents from the filtrate. A yield of4.1 g. of 4,5-dibromo-o-phenylenediamine was obtained.

4.5-Dibromo-o-phenylaminediamine was cyclized to the correspondingquinoxaline carboxylic acid ester by the procedure of Example 1utilizing 4.1 g. of the diamine and 2.7 g. of diethyl ketomalonate in 75ml. of anhydrous ethanol. Ethyl6,7-dibromo-3,4-dihydro-3-oxo-2-quinoxaline carboxylate thus preparedmelted at 235°-236° C. (yield=3.9 g.).

EXAMPLE 3 Preparation of Ethyl6(7)-Chloro-7(6)-bromo-3,4-dihydro-3-oxo-2-quinoxaline Carboxylate.

3-Chloro-4-bromoacetanilide prepared by the procedure of Example 2 wasnitrated in 18 M aqueous sulfuric acid with propyl nitrate at 0° C.according to the procedure of Example 2. The product of the reaction wasworked up by adding it to a mixture of ice and water with stirring. Ayellow powder comprising 3-chloro-4-bromo-6-nitroacetanilideprecipitated and was collected by filtration. Recrystallization fromethanol gave crystals melting at 128°-130° C.; yield=38 g.

3-Chloro-4-bromo-6-nitroacetanilide was hydrolyzed to the free amine bythe process of Example 2. Reduction of 5 g. of3-chloro-4-bromo-6-nitroaniline thus formed with Raney nickel by theprocedure of Example 2 yielded 3.35 g. of4(5)-chloro-5(4)-bromo-o-phenylenediamine. A 1.66 g. portion of thediamine were reacted with 0.85 g. of diethyl ketomalonate by refluxingin a mutual solvent for 2.5 hours. Yellow, needle-like crystalscomprising ethyl 6(7)-chloro-7(6)-bromo-3,4-dihydro-3-oxo-2-quinoxalinecarboxylate precipitated and were collected by filtration; mp=185°-195°C.; yield=111 g.

In the above reaction scheme, when each X represents a different group,the product of the reaction of the substituted o-phenylenediamine anddiethyl mesoxalate produces a mixture of compounds as seen in Example 3.A reaction procedure for the unequivocal synthesis of a compound comingwithin the scope of this invention in which the X's are different is setforth in Reaction Scheme 2 below: ##STR4## in which one X is Br and theother Cl.

According to Reaction Scheme 2, a 6-nitro-3,4-dihaloaniline (IV) isreacted with ethyl malonyl chloride (or other alkyl malonyl halide) togive the corresponding ethyl malonyl amide derivative on the anilinenitrogen (V). A base catalyzed annelation using sodium ethoxide at 0° C.yields the quinoxaline N¹ -oxide (VI), treatment of which withphosphorus trichloride in tetrahydrofuran (THF) at ambient temperatureproduces unambiguously a 6,7-substituted-3,4-dihydro-3-oxo-2-quinoxalinecarboxylate, ethyl ester (VII). This procedure is illustrated in Example4 below.

EXAMPLE 4 Preparation of Ethyl6-Chloro-7-bromo-3,4-dihydro-3-oxo-2-quinoxaline Carboxylate.

Five grams of 3-chloro-4-bromo-6-nitroaniline were dissolved in 150 ml.of benzene. Five grams of the acid chloride of monoethyl malonate wereadded with stirring under a nitrogen atmosphere. The reaction mixturewas heated to refluxing temperature overnight. Thin-layer chromatographyindicated that the reaction was essentially complete at this time. Thereaction mixture was cooled and the benzene removed by evaporation invacuo. The residue containing N-ethoxycarbonylacetyl3-chloro-4-bromo-6-nitroaniline formed in the above reaction, wasrecrystallized from anhydrous ethanol to yield fluffy yellow crystalsmelting at 119°-121° C.

Sodium ethylate was prepared under anhydrous conditions from 35 ml. ofanhydrous ethanol and 1 g. of sodium in a nitrogen atmosphere. Themixture was stirred until the sodium was dissolved completely afterwhich time the mixture was chilled to about 0° C. N-ethoxycarbonylacetyl3-chloro-4-bromo-6-nitroaniline was added and the resulting mixture wasstirred at 0° C. for about 3 hours. The reaction was then quenched byadding it to 300 ml. of 1 N aqueous hydrochloric acid at 0° C. Thisaqueous mixture was stirred until a solid precipitate formed. Theprecipitate was separated by filtration, dried, and the filter cake wasrecrystallized from anhydrous ethanol. Ethyl6-chloro-7-bromo-3-oxo-2-quinoxaline carboxylate N-oxide thus preparedmelted at 219°-221° C.; yield=2 g.

Analysis Calc.: C, 38.01; H, 2.32; N, 8.06;

Found: C, 37.79; H, 2.35; N, 8.24

One gram of ethyl 6-chloro-7-bromo-3,4-dihydro-3-oxo-2-quinoxalinecarboxylate N-oxide was dissolved in 50 ml. of THF. Six ml. ofphosphorus trichloride were added and the resulting mixture heatedgently at refluxing temperature overnight. The reaction mixture waspoured into 500 ml. of an ice-water mixture. A solid, comprising ethyl6-chloro-7-bromo-3,4-dihydro-3-oxo-2-quinoxaline carboxylate formed inthe above reaction, was separated by filtration; melting point=203°-205°C.; yield=0.6 g.

While it is easier to have an ethyl or methyl ester in the final product(I) by starting with the desired diethyl or dimethyl mesoxalate, it ispossible to hydrolyze the ester produced and reesterify the resultingacid with the other alcohol as illustrated below.

EXAMPLE 5 Hydrolysis of Ethyl6,7-Dichloro-3,4-dihydro-2-oxo-3-quinoxaline Carboxylate.

One gram of ethyl 6,7-dichloro-3,4-dihydro-2-oxo-3-quinoxalinecarboxylate was dissolved in a mixture of 25 ml. of isopropanol and 75ml. of water. Five grams of potassium hydroxide were added and theresulting mixture heated to reflux temperature for 5 minutes. The hotreaction mixture was decolorized with activated charcoal and filtered.The filtrate was acidified with 12 N aqueous hydrochloric acid.Needle-like yellow crystals precipitated comprising6,7-dichloro-2-oxo-3-quinoxaline carboxylic acid formed in the abovehydrolysis. The acid was collected by filtration.

EXAMPLE 6 Preparation of esters

6,7-Dichloro-3,4-dihydro-3-oxo-2-quinoxaline carboxylic acid preparedfrom the corresponding ethyl ester by the procedure of Example 5 wasesterified with methanol according to the following procedure: Two gramsof the free acid and 20 ml. of anhydrous methanol were mixed with acatalytic quantity of boron trifluoride etherate dissolved in methanol.The mixture was heated to refluxing temperature for 60 hours after whichtime it was cooled and filtered. Methyl6,7-dichloro-3,4-dihydro-3-oxo-2-quinoxaline carboxylate separated andwas isolated by filtration; weight=153 g.; color=greenish yellow;mp=258°-260° C.

The hindered amine component of my novel antiviral combination can beprepared by methods available in the art.

The synergistic antiviral effects of the combination of a quinoxalineand a hindered amine is illustrated by the following experiments. Eachcomponent, the quinoxaline and the hindered amine, were testedindividually at a series of dose levels against Ann Arbor or Maryland Bstrains of influenza virus. Combinations of the two drugs using a doselevel corresponding to one already used in determining the efficacy ofthe individual components was employed. The general procedure is thatset forth in Redman et al., Antimicrobial Agents and Chemotherapy, 497(1966). The mean day of death for the treated and untreated mice wasrecorded and the number of survivors out of the total number of mice ateach dose level also recorded. In addition a survival index wascomputed. The survival index is a composite measure of effectivenessincorporating both time of death, the number of survivors into a singlevariable in accordance with the above paper by Redman et al. In tables 1and 2 which follow, the drugs were given by the intraperitoneal route.The dosage was administered 24 hours and 4 hours prior to inoculationwith the virus and 24 and 48 hours after inoculation. Groups of 18 micewere used at each dose level and there was a control group which wasgiven only the pharmaceutical extending medium for each determination.White swiss (McAllister strain) females weighing 11 to 13 grams wereused. Statistically-significant data is indicated in the tables in eachinstance by an asterisk. For each of following the data in the tables,each compound employed in the experiment has been given a letteraccording to the following chart

A. ethyl 6,7-dibromo-3,4-dihydro-3-oxoquinoxaline-2-carboxylate,

B. ethyl-6,7-dichloro-3,4-dihydro-3-oxoquinoxaline-2-carboxylate,

M. 1-aminoadamantane hydrochloride,

N. 3-methyl-1-adamantane hydrochloride,

O. dl-cyclooctylamine,

P. 1(1-adamantyl)-1-amino ethane, and

R. 2-aminonorbornane.

In each table that follows the strain of virus is indicated at the headof the table. Column one gives the letter assigned to a given compound,column 2 the dose in mg./kg., column 3, the survival index at that doselevel, column 4, the median day of death, column 5, the number ofsurvivors and column 6 the number of animals. Table 3 gives similarinformation for the administration of combinations of adamantylaminehydrochloride and ethyl 6,7-dibromo-3,4-dihydro-3-oxoquinoxalinecarboxylate administered to mice by the oral route at a series of doselevels at the same time intervals before and after inoculation of themice with the Ann Arbor strain of influenza virus as for Tables 1 and 2.

                  TABLE 1                                                         ______________________________________                                        Ann Arbor Strain - I.P.                                                                                 Medium                                                      Dose in  Survival Day    Number Survivors                             Compound                                                                              mg/kg    Index    of Death                                                                             Number Animals                               ______________________________________                                        M       40       5.26*    9.1    10/17                                        M       80       5.67*    8.8    14/17                                        A       40       2.79     7.9    4/18                                         A       80       3.62     8.6    4/18                                         M + A   40 + 40  6.17*    8.0    17/18                                        Control 0        2.93     8.1    7/35                                         M       40       6.20*    9.0    7/17                                         A       80       5.74*    8.9    3/18                                         M + A   20 + 40  6.70*    9.1    11/18                                        M + A   10 + 20  5.14*    8.5    5/18                                         M + A   5 + 10   4.73*    8.2    5/18                                         Control 0        2.46     7.5    1/36                                         M       40       7.23*    9.7    7.18                                         M       80       6.52*    9.2    9.18                                         A       40       4.82*    8.9    1/18                                         A       80       6.25*    9.4    4/18                                         M + A   40 + 40  6.78*    9.0    13/18                                        M + A   20 + 20  7.04*    9.5    7/18                                         M + A   10 + 10  6.27*    9.3    5/18                                         Control 0        2.21     8.1    2/36                                         M       80       6.02*    8.9    8/17                                         M + A   10 + 20  6.86*    8.5    14/18                                        M + A   20 + 60  7.45*    9.7    12/18                                        M + A   30 + 60  7.38*    9.3    14/18                                        Control 0        2.60     8.1    1/36                                         N       25       4.07*    7.9    4/18                                         N       50       5.53*    9.1    5/18                                         A       40       4.86*    8.7    2/18                                         A       80       5.51*    9.3    3/18                                         N + A   25 + 40  5.97*    9.0    8/18                                         Control 0        2.39     7.6    0/36                                         N       20       2.89     7.9    1/18                                         N       40       3.69     8.2    1/18                                         A       40       3.94*    8.2    1/18                                         A       80       4.78*    8.7    0/18                                         N + A   20 + 40  6.13*    8.8    9/18                                         Control 0        2.36     7.6    2/36                                         0       40       4.05*    8.4    1/18                                         0       80       4.05*    8.3    2/18                                         A       40       3.36     8.0    2/18                                         A       80       4.06*    8.4    1/18                                         0 + A   40 + 40  5.90*    8.8    6/18                                         Control 0        2.51     7.6    1/36                                         M       40       5.40*    9.1    6/18                                         M       80       5.69*    9.1    9.18                                         B       40       3.27     8.3    3/18                                         B       80       3.07     8.3    3/18                                         M + B   40 + 40  5.85*    7.8    13/18                                        Control 0        1.71     7.7    4/36                                         M       40       3.85     8.7    2/18                                         M       80       4.42*    8.7    6/17                                         B       40       2.87     8.3    1/17                                         B       80       3.24     8.5    1/18                                         M +  B  40 + 40  5.68*    9.4    9/18                                         Control 0        1.76     7.7    0/36                                         O       40       5.09*    8.8    2/18                                         O       80       5.89*    8.9    3/18                                         A       40       4.12*    8.1    1/18                                         A       80       4.97*    8.4    1/18                                         O + A   40 + 40  6.07*    9.0    4/18                                         Control 0        2.38     7.3    0.36                                         P       40       2.46     7.4    0/18                                         P       80       2.61     7.5    0/18                                         A       40       4.65*    8.3    0/18                                         A       80       4.51*    8.2    1/18                                         P + A   40 + 40  4.18*    7.6    5/18                                         Control 0        2.31     7.3    1/36                                         R       40       5.91*    9.5    7/18                                         R       80       7.20*    10.0   14/18                                        A       40       2.86     7.9    4/18                                         A       80       3.98     8.9    3/18                                         R + A   40 + 40  7.30*    10.0   16/18                                        Control 0        1.83     7.6    1/36                                         O       80       5.19*    7.6    7/17                                         A       40       4.41     8.7    2/17                                         A       80       4.95     8.8    4/18                                         O + A   40 + 40  5.54*    7.9    7/17                                         Control 0        2.75     8.1    1/36                                         P       40       2.36     8.0    1/18                                         P       80       3.21     7.8    1/18                                         A       40       5.99*    8.9    1/18                                         A       80       6.26*    8.9    3/18                                         P + A   40 + 40  4.27*    8.2    2/18                                         Control 0        2.24     7.6    0/36                                         ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Maryland B Strain - I.P.                                                                                Medium                                                      Dose in  Survival Day    Number Survivors                             Compound                                                                              mg/kg    Index    of Death                                                                             Number Animals                               ______________________________________                                        M       40       2.39     8.2    0/18                                         A       80       4.44*    8.7    4/18                                         M + A   10 + 20  2.41     8.3    0/18                                         M + A   20 + 40  4.56*    8.5    5/18                                         M + A   40 + 80  4.79*    8.8    3/17                                         Control          2.20     8.2    1/36                                         ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Ann Arbor Strain - Oral                                                                                 Medium                                                      Dose in  Survival Day    Number Survivors                             Compound                                                                              mg/kg    Index    of Death                                                                             Number Animals                               ______________________________________                                        M       80       4.97*    8.9    5/18                                         M + A   10 + 20  3.06*    8.3    2/18                                         M + A   20 + 40  5.12*    8.5    7/18                                         M + A   30 + 60  6.03*    8.9    9/18                                         M + A   40 + 80  6.01*    8.9    9/17                                         Control 0        2.83     8.4    2.35                                         ______________________________________                                    

The experimental results set forth in Tables 1-3 above, showing asynergistic effect between a quinoxaline of formula I and a hinderedamine, involve protocols whereby the combination of drugs isadministered either by the intraperitoneal route or orally. I preferthat the drugs be administered orally. For oral administration, a mostadvantageous mode of administration is by the use of gelatin capsules.For this purpose, a quantity of a quinoxaline ester according to formulaI above and a quantity of a hindered amine preferably as an acidaddition salt with a strong acid such as hydrochloric acid are mixed andthe mixture diluted with a pharmaceutically acceptable extending mediumsuch as starch. After thoroughly mixing, the mixture is loaded intoempty gelatin capsules such that each capsule contains from 50 to 100mg. of the acid addition salt of the hindered tertiary amine and from 50to 100 mg. of the quinoxaline. Since it is known that the esters areslowly hydrolyzed in the presence of amine hydrochlorides andatmospheric moisture, one of the two ingredients, either the quinoxalineor the amine hydrochloride, should be coacervated with, for example,ethyl cellulose, shellac or an enteric coating composed of for examplepolyvinylacetatephthalate or hydroxypropylmethylcellulose phthalate. Theteaching of U.S. Pat. No. 4,044,125 for inhibiting the hydrolysis of theester, acetylsalicyclic acid in the presence of a hydrochloride salt,d-propoxyphene hydrochloride, can be followed in which the compositionis stabilized by the addition of the hydrochloride salt of an aminoacid.

The novel drug combinations of this invention have the followingdesirable properties: The combination is more active in combating viralinfections and suppressing viral growth in mammals in vivo than iseither ingredient by itself, as evidenced by the side-by-side tests setforth in the above tables. There is not only a higher degree ofprotection but also a higher therapeutic ratio at the low drug levelsthan for each of the drugs by itself. There are also fewer side effectswith the combination since the drugs act synergistically only in theireffect against influenza virus and not in producing side effects. Thesynergistic combination is active against both A and B strains ofinfluenza virus as exemplified by the Ann Arbor and Maryland "B" strainsincluded in the above experimental work. Thirdly the combination has alow toxicity. Lastly and most importantly, the combination is activeupon oral administration.

I claim:
 1. A method of suppressing influenza virus infections inmammals which comprises orally administering to a mammal exposed toinfluenza virus a virus-suppressing amount of a combination containingfrom 0.5-5 mg./kg. of ethyl 6,7-bromo-4-dihydro-3-oxoquinoxalinecarboxylate and from 0.3-3 mg./kg. of 1-(1-adamantyl)-1-aminoethane.